SUMMARY An important mechanism of gene expression regulation is dynamically regulated, and possibly reversible, nucleotide modifications in mRNA. These modifications can have marked effects on mRNA stability, translation, and other aspects of mRNA metabolism. We had a founding role in this field by developing the technology for transcriptome-wide mapping of N6-methyladenosine (m6A). Our mapping study provided the first evidence that m6A could be dynamically regulated, and potentially impart new functions in mRNA. We recently showed that acute myeloid leukemia (AML) cells exhibit elevated levels of METTL3 and METTL14, the heterodimer that acts as the m6A-forming methyltransferase. We found that m6A promotes self-renewal in AML and in CD34+ stem cells, and depletion of m6A triggers a differentiation program. Thus, m6A has critical roles in hematopoietic differentiation at specific stages of development, and this process is deregulated in AML. Therefore, precise characterization of these stage-specific patterns of m6A at a transcriptome-wide level is critical to understand how m6A affects developmental transitions. Developing new methods to map m6A in the rare cell populations relevant to hematopoiesis and AML would help to reveal how this epitranscriptomic modification is critical for the regulation and deregulation of differentiation seen in AML, and possibly other cancers. Additionally, the effects of m6A are largely thought to reflect the actions of specific ?reader? proteins, which bind m6A in mRNA to affect its fate in cells. The major readers are YTHDC1 in the nucleus, and the YTHDF family in the cytoplasm, which comprise three nearly identical paralogs, and which may have redundant functions. In order to significantly advance our understanding of the role of m6A in AML, the specific aims of this proposal are: (1) To visualize and map m6A in mRNA in a cell-type specific manner. Here we describe the development of methods for detecting and mapping m6A in a cell type-specific manner and their application to understand m6A dynamics in hematopoiesis and AML. (2) To define the functional requirement for the m6A reader YTHDC1 in normal blood cells and in AML. Based on a genome-wide screen and our preliminary data, YTHDC1 is a strong candidate for the reader that may mediate major aspects of the effect of m6A in AML. Here we assess the functional role for YTHDC1 in both normal and malignant hematopoiesis using human cord blood cells, AML cell lines and primary AML patients. (3) To determine the roles and regulation of the YTHDF cytosolic m6A readers on mRNA fate. The YTHDF proteins appear to be the major regulators of m6A mRNAs in the cytosol. We will determine how YTHDF proteins are regulated to mediate their m6A-mRNA destabilizing effects and if YTHDF proteins influence cellular differentiation and proliferation in cancer cell lines and in AML. Overall, our project will develop new enabling technologies for studying m6A in cancer and test mechanisms by which the m6A readers contribute to cancer progression.